Multidimensional protein identification technology (MudPIT) has been developed to identify proteins in complex mixtures without prior separation by gel electrophoresis. Enzymatically digested proteins are identified using multidimensional liquid chromatography coupled with automated interpretation of tandem mass spectrometry. An integrated, microcapillary variation of the chromatography and electrospray ionization source gives the process the equivalent sensitivity as a silver-stained polyacrylamide gel. The technique was validated using the yeast 78 protein 80S ribosomal complex. Using this process, we have identified a novel component of the yeast 40S-ribosome subunit and found the homologous protein in the human 40S subunit.